Plastids can be classified as follows: chloroplasts for photosynthesis, leucoplasts lacking in pigments, and chromoplasts involved in the coloring of fruits and flowers. A single plant cell may have up to 200 plastids, and a single plastid may have 100 copies of its genome; thus a single cell may have 10,000˜50,000 copies of plastid genome. On the other hand, a nucleus of a plant cell carries only 1 or 2 copies of the genome. Thus, one can produce a heterologous protein using plastid transformation of a gene encoding the heterologous protein about 10,000-fold more effectively than a nuclear transformation
Most researches on plastid transformation have been conducted in tobacco (Nicotiana tabacum). Successful transformations were reported from such organisms as Arabidopsis, potato and tomato, but plants other than tobacco are generally known to suffer from poor transformation efficiencies. The reason why the transformation efficiencies are low seems that the transformation of plastids is carried out inadequately and that long terms and complicated operations are required for selection of transformants as a result. It is possible to achieve highly efficient transformation in tobacco since the natures of transformation have been well known through a number of researches thereon.
Under this rationale, a few plastid transformation methods were recently developed to impart new traits to plants by introducing a heterologous gene (Svab, Z., Hajdukiewicz, Maliga, P., Proc. Natl. Acad. Sci., 87, pp 8526˜8530, 1990; Staub, J. M. et al, Nature Biotechnol., 18, pp 333˜338, 2000). Such transformation procedures consist of broadly two steps: plastid transformation and selection of transformants.
In order to overcome the problems of low transformation efficiency and long term for selection required for producing homoplasmy in plastid transformation and to develop a simple and efficient procedure for plastid transformation, the present inventors had provided a method of plastid transformation with high rate of homologous recombination and transformation which comprises constructing a vector for plastid transformation comprising a heterologous gene and a selection marker gene, performing plastid transformation by introducing the vector into a plant in which a heterologous recombinase incorporated in a nucleus is designed to translocate into plastids and selecting a transformant according to expression level of the selection marker gene (KR 2002-00218A).
The production of recombinant proteins has been mostly carried out in the microbial and animal cell systems However, researches focused on the production of a recombinant protein using plant and plant cell culture system have been actively in progress. Agrobacterium-mediated transformation is mainly used for introducing heterologous genes into plant cells. Unlike microbial systems, plant expression systems do not suffer from the lack of post-translational modification, which can raise value of recombinant proteins produced in plant system. In addition, plant systems have the advantages of less complicated culture condition and use of inexpensive media-compositions compared to animal systems, and less infection from animal viruses and toxins (Doran, P. M. Current Opinion in Biotechnology, 11, pp 199˜204, 2000). Currently a number of researches have been actively in progress to produce the recombinant protein using the plant cell culture system, and the following model proteins are noteworthy: □-glucuronidase (Kutara, H., Takemura, T., Furusaki, S., Kado, C. I., J. Ferment. Bioeng., 86, pp 317˜323, 1998), antibodies (LaCount, W., An, G., Lee, J. M., Biotechnology Letters, 19, pp 93˜96, 1997), interleukins (Magnuson, N. et al., Protein Expre. Purif., 13, pp 45˜52, 1998), ricin (Sehnki, P. C., Ferl, R. J., Protein Expr. Purif., 15, pp 188˜195, 1999), and □1-antitrypsin (Terashima, M. et al, Appl. Microbiol. Biotechnol., 52, pp 516˜523, 1999).
Tobacco suspension cells or hairy roots are mainly used as host for plant expression systems due to facileness of transformation, relative ease of fluorescence detection and rapid growth rate thereof (Wongsamuth, R., Doran. P. M., Biotechnol. Bioeng., 54(5): 401˜415, 1997). In addition, other plants may be used as hosts depending on the choice of a promoter for expressing the recombinant protein. For example, a rice cell culture system can be used, which uses a promoter that senses the depletion in sucrose levels (Terashima, M. et al., Appl. Microbiol. Biotechnol., 52, pp 516˜523, 1999).
The promoter/terminator pairs used in the prior arts for chloroplast transformation of tobacco are the promoters and terminators isolated from the same plant species. Such combinations, however, promote another homologous recombination between the terminator and the corresponding sequence in the original chloroplast genome of tobacco in addition to the first homologous recombination, producing plants with anomalies in the chloroplast genome (Staub, J. M., Maliga, P., Proc. Natl. Acad. Sci., 91, 7468˜7472, 1994; Svab, Z., Maliga, P., Proc. Natl. Acad. Sci., 90, 913˜917, 1993). The present inventors have confirmed that when plastid transformation vectors carrying Prrn/psbAT, a typical promoter/terminator pair were used, 100% of the transformants underwent a second recombination and that 50% of them had anomalous plastid whose genomes consist mostly of small subgenomes arising from the second recombination. In such cases, it was found that stable maintenance and expression of the incorporated heterologous genes were not guaranteed.